RESUMEN
Extracellular matrix remodeling mechanisms are understudied in cardiac development and congenital heart defects. We show that matrix-degrading metalloproteases ADAMTS1 and ADAMTS5, are extensively co-expressed during mouse cardiac development. The mouse mutants of each gene have mild cardiac anomalies, however, their combined genetic inactivation to elicit cooperative roles is precluded by tight gene linkage. Therefore, we coupled Adamts1 inactivation with pharmacologic ADAMTS5 blockade to uncover stage-specific cooperative roles and investigated their potential substrates in mouse cardiac development. ADAMTS5 blockade was achieved in Adamts1 null mouse embryos using an activity-blocking monoclonal antibody during distinct developmental windows spanning myocardial compaction or cardiac septation and outflow tract rotation. Synchrotron imaging, RNA in situ hybridization, immunofluorescence microscopy and electron microscopy were used to determine the impact on cardiac development and compared to Gpc6 and ADAMTS-cleavage resistant versican mutants. Mass spectrometry-based N-terminomics was used to seek relevant substrates. Combined inactivation of ADAMTS1 and ADAMTS5 prior to 12.5 days of gestation led to dramatic accumulation of versican-rich cardiac jelly and inhibited formation of compact and trabecular myocardium, which was also observed in mice with ADAMTS cleavage-resistant versican. Combined inactivation after 12.5 days impaired outflow tract development and ventricular septal closure, generating a tetralogy of Fallot-like defect. N-terminomics of combined ADAMTS knockout and control hearts identified a cleaved glypican-6 peptide only in the controls. ADAMTS1 and ADAMTS5 expression in cells was associated with specific glypican-6 cleavages. Paradoxically, combined ADAMTS1 and ADAMTS5 inactivation reduced cardiac glypican-6 and outflow tract Gpc6 transcription. Notably, Gpc6-/- hearts demonstrated similar rotational defects as combined ADAMTS inactivated hearts and both had reduced hedgehog signaling. Thus, versican proteolysis in cardiac jelly at the canonical Glu441-Ala442 site is cooperatively mediated by ADAMTS1 and ADAMTS5 and required for proper ventricular cardiomyogenesis, whereas, reduced glypican-6 after combined ADAMTS inactivation impairs hedgehog signaling, leading to outflow tract malrotation.
Asunto(s)
Proteína ADAMTS1 , Proteína ADAMTS5 , Glipicanos , Corazón , Proteolisis , Versicanos , Animales , Ratones , Versicanos/metabolismo , Versicanos/genética , Proteína ADAMTS5/metabolismo , Proteína ADAMTS5/genética , Proteína ADAMTS1/metabolismo , Proteína ADAMTS1/genética , Glipicanos/metabolismo , Glipicanos/genética , Corazón/crecimiento & desarrollo , Ratones Noqueados , Regulación del Desarrollo de la Expresión Génica , Cardiopatías Congénitas/genética , Cardiopatías Congénitas/metabolismo , Cardiopatías Congénitas/patologíaRESUMEN
In the study of tumorigenesis, the involvement of molecules within the extracellular matrix (ECM) is crucial. ADAMTSs (A Disintegrin and Metalloproteinase with Thrombospondin motifs), a group of secreted proteases known for their role in ECM remodeling, were primarily considered to be extracellular proteases. However, our research specifically detected ADAMTS-1, a member of this family, predominantly within the nucleus of mammary cells. Our main objective was to understand the mechanism of ADAMTS-1 translocation to the nucleus and its functional significance in this cellular compartment. Our investigation uncovered that nuclear ADAMTS-1 was present in cells exhibiting an epithelial phenotype, while cells of mesenchymal origin contained the protease in the cytoplasm. Moreover, disruption of ADAMTS-1 secretion, induced by Monensin treatment, resulted in its accumulation in the cytoplasm. Notably, our research indicated that alterations in the secretory pathways could influence the protease's compartmentalization. Additionally, experiments with conditioned medium from cells containing nuclear ADAMTS-1 demonstrated its internalization into the nucleus by HT-1080 cells and fibroblasts. Furthermore, heightened levels of ADAMTS-1 within the ECM reduced the migratory potential of mesenchymal cells. This highlights the potential significance of nuclear ADAMTS-1 as a critical component within the tumor microenvironment due to its functional activity in this specific cellular compartment.
Asunto(s)
Proteína ADAMTS1 , Movimiento Celular , Núcleo Celular , Matriz Extracelular , Trombospondinas , Humanos , Proteína ADAMTS1/genética , Proteína ADAMTS1/metabolismo , Carcinogénesis/metabolismo , Endopeptidasas/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Trombospondinas/metabolismo , Microambiente Tumoral , Núcleo Celular/metabolismoRESUMEN
Non-small cell lung cancer (NSCLC) accounts for approximately 80% of all lung cancers. Identifying key molecular targets related to the initiation, development, and metastasis of lung cancer is important for its diagnosis and target therapy. The ADAMTS families of multidomain extracellular protease enzymes have been reported to be involved in many physiological processes. In this study, we found that ADAMTS1 was highly expressed in NSCLC tissues, which promoted cell proliferation, migration, invasion, and epithelial to mesenchymal transition (EMT) of NSCLC cells. In the NSCLC tumor metastasis model involving nude mice, overexpression of ADAMTS1 promoted EMT and lung metastasis of tumor cells. Moreover, ADAMTS1 positively regulated TGF-ß expression, and TGF-ß was highly expressed in NSCLC tumor tissues. si-TGF-ß or inhibition of TGF-ß expression through the short peptide KTFR on ADAMTS1 protein could reverse the oncogenic effects of ADAMTS1 on lung cancer cells. Taken together, ADAMTS1 functioned as an oncogene in NSCLC cells by promoting TGF-ß expression, indicating that ADAMTS1 has important regulatory roles in the progression of NSCLC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , MicroARNs , Animales , Ratones , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/patología , Transición Epitelial-Mesenquimal/genética , Factor de Crecimiento Transformador beta/metabolismo , Ratones Desnudos , Proteína ADAMTS1/genética , Proteína ADAMTS1/metabolismo , MicroARNs/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Regulación Neoplásica de la Expresión GénicaRESUMEN
A disintegrin-like and metalloproteinase with thrombospondin type 1 motifs (ADAMTS1) is a protease involved in fertilization, cancer, cardiovascular development, and thoracic aneurysms. Proteoglycans such as versican and aggrecan have been identified as ADAMTS1 substrates, and Adamts1 ablation in mice typically results in versican accumulation; however, previous qualitative studies have suggested that ADAMTS1 proteoglycanase activity is weaker than that of other family members such as ADAMTS4 and ADAMTS5. Here, we investigated the functional determinants of ADAMTS1 proteoglycanase activity. We found that ADAMTS1 versicanase activity is approximately 1000-fold lower than ADAMTS5 and 50-fold lower than ADAMTS4 with a kinetic constant (kcat/Km) of 3.6 × 103 M-1 s-1 against full-length versican. Studies on domain-deletion variants identified the spacer and cysteine-rich domains as major determinants of ADAMTS1 versicanase activity. Additionally, we confirmed that these C-terminal domains are involved in the proteolysis of aggrecan as well as biglycan, a small leucine-rich proteoglycan. Glutamine scanning mutagenesis of exposed positively charged residues on the spacer domain loops and loop substitution with ADAMTS4 identified clusters of substrate-binding residues (exosites) in ß3-ß4 (R756Q/R759Q/R762Q), ß9-ß10 (residues 828-835), and ß6-ß7 (K795Q) loops. This study provides a mechanistic foundation for understanding the interactions between ADAMTS1 and its proteoglycan substrates and paves the way for development of selective exosite modulators of ADAMTS1 proteoglycanase activity.
Asunto(s)
Proteína ADAMTS1 , Animales , Ratones , Proteína ADAMTS1/química , Proteína ADAMTS1/metabolismo , Proteína ADAMTS4/metabolismo , Proteína ADAMTS5/metabolismo , Agrecanos/metabolismo , Versicanos/metabolismoRESUMEN
BACKGROUND: Glioma is the most common intracranial malignancy in adults with a high degree of malignancy and poor prognosis, which is largely attributed to the existence of glioma stem cells (GSCs). Previous evidence indicated that the matrix metalloproteinase ADAMTS1 was implicated in the process of tumor invasion, but the involvement of ADAMTS1 in glioma malignant invasion remains poorly understood. METHODS: The expression and prognosis values of ADAMTS1 were investigated in patients with glioma based on ONCOMINE and GEPIA databases. ADAMTS1 expression of different malignancy grade tissues was determined by immunohistochemistry. The effects of ADAMTS1 on cell proliferation and invasion were determined by clone formation assay and Transwell migration assay. The animal experiment was performed in an intracranial orthotopic xenograft model by knockout of ADAMTS1. Stemness properties and Notch1-SOX2 pathway were examined in stable ADAMTS1 knockdown GSCs. RESULTS: The expression levels of ADAMTS1 were significantly higher in glioma tissues and significantly correlated with the grade of malignancy and prognosis of glioma. Elevated ADAMTS1 expression was associated with SOX2, N-cadherin and the resistance of chemoradiotherapy of glioma patients. ADAMTS1 knockout suppressed the intracranial orthotopic xenograft growth and prolonged the survival of xenograft mice in vivo. Mechanistically, we found a blockade of the migration and invasiveness of GSCs and the expression levels of Notch1 and SOX2 in absence of ADAMTS1. CONCLUSION: As a biomarker for prediction of prognosis, ADAMTS1 may affect the invasive phenotype of GSCs by regulating Notch1-SOX2 signaling pathway, thereby promoting the invasive growth of glioma.
Asunto(s)
Neoplasias Encefálicas , Glioma , Humanos , Ratones , Animales , Pronóstico , Línea Celular Tumoral , Glioma/patología , Neoplasias Encefálicas/patología , Transducción de Señal , Proliferación Celular/genética , Células Madre Neoplásicas/patología , Regulación Neoplásica de la Expresión Génica , Proteína ADAMTS1/genética , Proteína ADAMTS1/metabolismoRESUMEN
Secreted protein acidic and rich in cysteine (SPARC), an extracellular matrix (ECM) protein, was recently shown to induce collagen deposition through the production of a disintegrin and metalloproteinase with thrombospondin type 1 motif (ADAMTS1) in the aging heart. ADAMTS1 regulates ECM turnover by degrading ECM components, and its excessive activation contributes to various pathological states, including fibrosis. The present study investigated the pathophysiological regulation and role of SPARC and ADAMTS1 in renal fibrosis using uninephrectomized rats treated with deoxycorticosterone acetate (DOCA, 40 mg/kg/week, subcutaneously) and salt (1% in drinking water). The administration of DOCA and salt gradually and significantly elevated systolic blood pressure during the 3-week treatment period, induced proteinuria, decreased creatinine clearance, and increased NADPH oxidase-derived superoxide production, malondialdehyde concentrations, and monocyte chemoattractant protein-1 and osteopontin expression in the kidneys. Glomerulosclerosis, fibrillar collagen deposition, and transforming growth factor-ß expression increased in a time-dependent manner, and SPARC and ADAMTS1 expression showed a similar pattern to these changes. The angiotensin II type-1 receptor blocker losartan suppressed the overexpression of SPARC and ADAMTS1, and an in vitro exposure to angiotensin II induced the production of both SPARC and ADAMTS1 in renal fibroblast NRK-49F cells. Knockdown of the SPARC gene with small interfering RNA reduced all forms (the 110-kDa latent and 87- and 65-kDa bioactive forms) of ADAMTS1 expression as well as collagen production. These results suggest that SPARC is induced by the renin-angiotensin system and may be a fibrogenic factor, at least in part, by producing ADAMTS1 in hypertensive renal disease.
Asunto(s)
Proteína ADAMTS1/metabolismo , Colágenos Fibrilares , Riñón , Losartán/farmacología , Osteonectina/metabolismo , Sistema Renina-Angiotensina , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Animales , Acetato de Desoxicorticosterona/farmacología , Proteínas de la Matriz Extracelular/metabolismo , Colágenos Fibrilares/biosíntesis , Colágenos Fibrilares/metabolismo , Fibrosis/metabolismo , Glomeruloesclerosis Focal y Segmentaria/etiología , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Glomeruloesclerosis Focal y Segmentaria/fisiopatología , Hipertensión/inducido químicamente , Hipertensión/metabolismo , Riñón/metabolismo , Riñón/patología , Mineralocorticoides/farmacología , Ratas , Sistema Renina-Angiotensina/efectos de los fármacos , Sistema Renina-Angiotensina/fisiología , Cloruro de Sodio Dietético/administración & dosificaciónRESUMEN
Hearing impairment (HI) is a common disorder of sensorineural function with a highly heterogeneous genetic background. Although substantial progress has been made in the understanding of the genetic etiology of hereditary HI, many genes implicated in HI remain undiscovered. Via exome and Sanger sequencing of DNA samples obtained from consanguineous Pakistani families that segregate profound prelingual sensorineural HI, we identified rare homozygous missense variants in four genes (ADAMTS1, MPDZ, MVD, and SEZ6) that are likely the underlying cause of HI. Linkage analysis provided statistical evidence that these variants are associated with autosomal recessive nonsyndromic HI. In silico analysis of the mutant proteins encoded by these genes predicted structural, conformational or interaction changes. RNAseq data analysis revealed expression of these genes in the sensory epithelium of the mouse inner ear during embryonic, postnatal, and adult stages. Immunohistochemistry of the mouse cochlear tissue, further confirmed the expression of ADAMTS1, SEZ6, and MPDZ in the neurosensory hair cells of the organ of Corti, while MVD expression was more prominent in the spiral ganglion cells. Overall, supported by in silico mutant protein analysis, animal models, linkage analysis, and spatiotemporal expression profiling in the mouse inner ear, we propose four new candidate genes for HI and expand our understanding of the etiology of HI.
Asunto(s)
Proteína ADAMTS1/genética , Carboxiliasas/genética , Pérdida Auditiva Sensorineural/genética , Proteínas de la Membrana/genética , Proteína ADAMTS1/química , Proteína ADAMTS1/metabolismo , Animales , Carboxiliasas/química , Carboxiliasas/metabolismo , Femenino , Genes Recesivos , Células Ciliadas Auditivas/metabolismo , Pérdida Auditiva Sensorineural/patología , Humanos , Masculino , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Ratones , Mutación , Linaje , Dominios ProteicosRESUMEN
AIM: To analyze the expression of ADAMTS-1, NF-?B, and STAT3 in human pleomorphic xanthoastrocytoma specimens, and their correlation with glioma advancement. MATERIAL AND METHODS: Pleomorphic xanthoastrocytoma tumor cell lines were treated with low and high doses of cytokines at 24 and 48 hours (h) to replicate the inflammatory environment. The effects of IL-1 were assessed with the scratch wound-healing assay, and the expression levels of ADAMTS-1, NF-?B, and STAT3 of the groups were determined by western blot analysis. RESULTS: Cytokine treatment significantly increased the migration of PXA glioma cells after scratching at 24h and 48h time points. Similarly, 10 and 30 ng/mL IL-1 induced 1.86 and 1.94 fold increases, respectively, in ADAMTS-1 expression after 24h, and 3 and 3.27 fold increases, respectively, after 48h, compared with the non-treatment control group.10 and 30 ng/mL IL-1 doses caused 2.5 and 2.6 fold increase, in NF-?B protein levels after 24h, and 3.16 and 3.41 fold increases after 48h, compared with the non-treatment group. The protein levels of STAT3 after 24h were 2.62 and 2.43 fold higher, and 3.78 and 3.84 fold higher after 48 hours, with 10 and 30 ng/mL IL-1, compared with the non-treatment group. CONCLUSION: The proliferation and progression of glioma cells were proportional to the increased expression levels of ADAMTS-1, NF-?B, and STAT3. Our findings indicate that the proteolytic function of ADAMTS-1 may be associated with the malignant transformation of low-grade gliomas.
Asunto(s)
Proteína ADAMTS1/metabolismo , Astrocitoma , Glioma , Transformación Celular Neoplásica , Citocinas , HumanosRESUMEN
Duchenne Muscular Dystrophy (DMD) is caused by mutations in the dystrophin gene, accompanied by aberrant extracellular matrix synthesis and muscle damage. ADAMTS1 metalloproteinase was reported increased in dystrophin-deficient mdx mouse. The aim of this study was to explore the role of ADAMTS1 in muscle function, fibrosis and damage, and respiratory function of mdx mice. 102 DMD patients and their mothers were included in this study. Multiplex ligation dependent probe amplification (MLPA) assay and Next-generation sequencing (NGS) were adopted to do genetic diagnosis. Dystrophin-deficient mdx mice were treated with anti-ADAMTS1 antibody (anti-ADAMTS1) for three weeks. The results showed that ADAMTS1 was increased in gastrocnemius muscle of mdx mice and serum of DMD patients. Anti-ADAMTS1 treatment increased Versican transcription but suppressed versican protein expression. Besides, we found anti-ADAMTS1 improved muscle strength, diaphragm and extensor digitorum longus muscles functions in mdx mice. Meanwhile, muscle fibrosis and damage were attenuated in anti-ADAMTS1 treated dystrophic mice. In summary, anti-ADAMTS1 antibody relieved muscle dysfunction and fibrosis in dystrophic mice. It is suggested that ADAMTS1 is a potential target for developing new biological therapies for DMD.
Asunto(s)
Proteína ADAMTS1/antagonistas & inhibidores , Anticuerpos Monoclonales/uso terapéutico , Músculo Esquelético/efectos de los fármacos , Distrofia Muscular de Duchenne/terapia , Proteína ADAMTS1/genética , Proteína ADAMTS1/inmunología , Proteína ADAMTS1/metabolismo , Animales , Modelos Animales de Enfermedad , Distrofina/genética , Fibrosis/terapia , Humanos , Masculino , Ratones , Ratones Endogámicos mdx , Proteínas Musculares/metabolismo , Fuerza Muscular/inmunología , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Distrofia Muscular de Duchenne/genética , ARN Mensajero/metabolismo , Versicanos/inmunologíaRESUMEN
Prostaglandins (PGs) are a class of fatty-acid derived hormones that are essential in ovulation of teleosts, but their exact role remains unknown. One putative target of PGs in ovulation is regulation of the expression of members of the A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS) family, which are implicated in follicular rupture. This study investigated the regulation of ADAMTS, other proteases, and their inhibitors in response to treatment with PGE2 or PGF2α. Four members of the ADAMTS family, ADAMTS1, ADAMTS5, ADAMTS9, and ADAMTS16 were shown to be expressed in the ovary of zebrafish, but only adamts1 was upregulated in full-grown follicles following treatment with PGE2. Inhibitors of the PG receptors EP1 and EP2 had no effect on PGE2-stimulated adamts1 expression, while treatment of full-grown follicles with both PGE2 and GW627368x, an inhibitor of EP4 function, prevented the PGE2-induced increase in adamts1 expression. Treatment of full-grown follicles with the maturation-inducing hormone 17α,20ß-dihydroxy-4-pregnen-3-one (17,20ß-P) in vitro had no effect on the expression of adamts1 mRNA. These findings suggest that expression of ADAMTS1 in zebrafish ovarian follicles is regulated by the prostaglandin PGE2 via the EP4 series prostaglandin receptor.
Asunto(s)
Ovario , Pez Cebra , Proteína ADAMTS1/metabolismo , Animales , Femenino , Folículo Ovárico/metabolismo , Ovulación/fisiología , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Pez Cebra/genéticaRESUMEN
White adipose tissue not only serves as a reservoir for energy storage but also secretes a variety of hormonal signals and modulates systemic metabolism. A substantial amount of adipose tissue develops in early postnatal life, providing exceptional access to the formation of this important tissue. Although a number of factors have been identified that can modulate the differentiation of progenitor cells into mature adipocytes in cell-autonomous assays, it remains unclear which are connected to physiological extracellular inputs and are most relevant to tissue formation in vivo. Here, we elucidate that mature adipocytes themselves signal to adipose depot-resident progenitor cells to direct depot formation in early postnatal life and gate adipogenesis when the tissue matures. Our studies revealed that as the adipose depot matures, a signal generated in mature adipocytes is produced, converges on progenitor cells to regulate the cytoskeletal protein MYH9, and attenuates the rate of adipogenesis in vivo.
Asunto(s)
Proteína ADAMTS1/genética , Adipocitos/metabolismo , Adipogénesis/genética , Tejido Adiposo Blanco/metabolismo , Homeostasis/genética , Cadenas Pesadas de Miosina/genética , Células Madre/metabolismo , Proteína ADAMTS1/metabolismo , Tejido Adiposo/metabolismo , Animales , Masculino , Ratones , Ratones Transgénicos , Cadenas Pesadas de Miosina/metabolismoRESUMEN
This study was designed to test whether the Cronobacter sakazakii infection-impaired contextual learning and memory are mediated by the activation of the complement system; subsequent activation of inflammatory signals leads to alternations in serotonin transporter (SERT). To test this, rat pups (postnatal day, PND 15) were treated with either C. sakazakii (107 CFU) or Escherichia coli OP50 (107 CFU) or Luria bertani broth (100 µL) through oral gavage and allowed to stay with their mothers until PND 24. Experimental groups' rats were allowed to explore (PNDs 31-35) and then trained in contextual learning task (PNDs 36-43). Five days after training, individuals were tested for memory retention (PNDs 49-56). Observed behavioural data showed that C. sakazakii infection impaired contextual-associative learning and memory. Furthermore, our analysis showed that C. sakazakii infection activates complement system complement anaphylatoxin (C5a) (a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS1)) and mitogen-activated protein kinase kinase1 (MEKK1). Subsequently, MEKK1 induces pro-inflammatory signals possibly through apoptosis signal-regulating kinase-1 (ASK-1), c-Jun N-terminal kinase (JNK1/3) and protein kinase B gamma (AKT-3). In parallel, activated nuclear factor kappa-light-chain-enhancer B cells (NF-κB) induces interleukin-6 (IL-6) and IFNα-1, which may alter the level of serotonin transporter (SERT). Observed results suggest that impaired contextual learning and memory could be correlated with C5a-mediated NF-κß and ASK1 pathways.
Asunto(s)
Aprendizaje por Asociación/fisiología , Activación de Complemento , Complemento C5a/fisiología , Cronobacter sakazakii/patogenicidad , Infecciones por Enterobacteriaceae/complicaciones , Discapacidades para el Aprendizaje/etiología , MAP Quinasa Quinasa Quinasa 5/fisiología , Trastornos de la Memoria/etiología , FN-kappa B/fisiología , Proteínas del Tejido Nervioso/fisiología , Serotonina/metabolismo , Transducción de Señal/fisiología , Proteína ADAMTS1/metabolismo , Animales , Animales Lactantes , Corteza Cerebral/metabolismo , Infecciones por Enterobacteriaceae/inmunología , Infecciones por Escherichia coli/complicaciones , Infecciones por Escherichia coli/inmunología , Regulación de la Expresión Génica/inmunología , Inflamación , Interferón-alfa/metabolismo , Interleucina-6/metabolismo , Quinasas Janus/metabolismo , Discapacidades para el Aprendizaje/inmunología , Discapacidades para el Aprendizaje/microbiología , Quinasa 1 de Quinasa de Quinasa MAP/metabolismo , Trastornos de la Memoria/inmunología , Trastornos de la Memoria/microbiología , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismoRESUMEN
ADAMTSs (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) are secreted proteases dependent on Zn2+/Ca2+, involved in physiological and pathological processes and are part of the extracellular matrix (ECM). Here, we investigated if ADAMTS-1 is required for invasion and migration of cells and the possible mechanism involved. In order to test ADAMTS-1's role in ovarian cancer cells (CHO, NIH-OVCAR-3 and ES2) and NIH-3 T3 fibroblasts, we modified the levels of ADAMTS-1 and compared those to parental. Cells exposed to ADAMTS-1-enriched medium exhibited a decline in cell migration and invasion when compared to controls with or without a functional metalloproteinase domain. The opposite was observed in cells when ADAMTS-1 was deleted via the CRISPR/Cas9 approach. The decline in ADAMTS-1 levels enhanced the phosphorylated form of Src and FAK. We also evaluated the activities of cellular Rho GTPases from cell lysates using the GLISA® kit. The Cdc42-GTP signal was significantly increased in the CRISPR ADAMTS-1 ES-2 cells. By a Förster resonance energy transfer (FRET) biosensor for Cdc42 activity in ES-2 cells we demonstrated that Cdc42 activity was strongly polarized at the leading edge of migrating cells with ADAMTS-1 deletion, compared to the wild type cells. As conclusion, ADAMTS-1 inhibits proliferation, polarization and migration.
Asunto(s)
Proteína ADAMTS1/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Proteína ADAMTS1/deficiencia , Proteína ADAMTS1/genética , Sistemas CRISPR-Cas/genética , Línea Celular , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular , Femenino , Quinasa 1 de Adhesión Focal/metabolismo , Factor de Crecimiento de Hepatocito/farmacología , Humanos , Fosforilación , ARN Guía de Kinetoplastida/metabolismo , Transducción de Señal , Familia-src Quinasas/metabolismoRESUMEN
The umbilical artery lumen closes rapidly at birth, preventing neonatal blood loss, whereas the umbilical vein remains patent longer. Here, analysis of umbilical cords from humans and other mammals identified differential arterial-venous proteoglycan dynamics as a determinant of these contrasting vascular responses. The umbilical artery, but not the vein, has an inner layer enriched in the hydrated proteoglycan aggrecan, external to which lie contraction-primed smooth muscle cells (SMC). At birth, SMC contraction drives inner layer buckling and centripetal displacement to occlude the arterial lumen, a mechanism revealed by biomechanical observations and confirmed by computational analyses. This vascular dimorphism arises from spatially regulated proteoglycan expression and breakdown. Mice lacking aggrecan or the metalloprotease ADAMTS1, which degrades proteoglycans, demonstrate their opposing roles in umbilical vascular dimorphism, including effects on SMC differentiation. Umbilical vessel dimorphism is conserved in mammals, suggesting that differential proteoglycan dynamics and inner layer buckling were positively selected during evolution.
Asunto(s)
Agrecanos/metabolismo , Miocitos del Músculo Liso , Arterias Umbilicales , Proteína ADAMTS1/metabolismo , Animales , Diferenciación Celular/fisiología , Femenino , Humanos , Ratones Transgénicos , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/fisiología , Parto/fisiología , Embarazo , Arterias Umbilicales/citología , Arterias Umbilicales/metabolismo , Arterias Umbilicales/fisiologíaRESUMEN
Cerebral cavernous malformations (CCMs) form following loss of the CCM protein complex in brain endothelial cells due to increased endothelial MEKK3 signaling and KLF2/4 transcription factor expression, but the downstream events that drive lesion formation remain undefined. Recent studies have revealed that CCM lesions expand by incorporating neighboring wild-type endothelial cells, indicative of a cell nonautonomous mechanism. Here we find that endothelial loss of ADAMTS5 reduced CCM formation in the neonatal mouse model. Conversely, endothelial gain of ADAMTS5 conferred early lesion genesis in the absence of increased KLF2/4 expression and synergized with KRIT1 loss of function to create large malformations. Lowering versican expression reduced CCM burden, indicating that versican is the relevant ADAMTS5 substrate and that lesion formation requires proteolysis but not loss of this extracellular matrix protein. These findings identify endothelial secretion of ADAMTS5 and cleavage of versican as downstream mechanisms of CCM pathogenesis and provide a basis for the participation of wild-type endothelial cells in lesion formation.
Asunto(s)
Proteína ADAMTS5/metabolismo , Hemangioma Cavernoso del Sistema Nervioso Central/etiología , Versicanos/metabolismo , Proteína ADAMTS1/metabolismo , Proteína ADAMTS4/metabolismo , Animales , Modelos Animales de Enfermedad , Endotelio Vascular/metabolismo , Femenino , Estudios de Asociación Genética , Hemangioma Cavernoso del Sistema Nervioso Central/embriología , Hemangioma Cavernoso del Sistema Nervioso Central/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Proteolisis , Sustancia Blanca/metabolismoRESUMEN
Prolificacy of most local goat breeds in China is low. Jining Grey goat is one of the most prolific goat breeds in China, it is an important goat breed for the rural economy. ASMT (acetylserotonin O-methyltransferase) and ADAMTS1 (ADAM metallopeptidase with thrombospondin type 1 motif) are essential for animal reproduction. Single nucleotide polymorphisms (SNPs) of ASMT and ADAMTS1 genes in the highly prolific breed (Jining Grey goats), medium prolific breed (Boer goats and Guizhou White goats) and low prolific breeds (Angora goats, Liaoning Cashmere goats and Inner Mongolia Cashmere goats) were detected by polymerase chain reaction-restriction fragment length polymorphism and sequencing. Two SNPs (g.158122T>C, g.158700G>A) of ASMT gene and two SNPs (g.7979798A>G, g.7979477C>T) of ADAMTS1 gene were identified. For g.158122T>C of ASMT gene, further analysis revealed that genotype TC or CC had 0.66 (p < 0.05) or 0.75 (p < 0.05) kids more than those with genotype TT in Jining Grey goats. No significant difference (p > 0.05) was found in litter size between TC and CC genotypes. The SNP (g.158122T>C) caused a p.Tyr298His change and this SNP mutation resulted in changes in protein binding sites and macromolecule-binding sites. The improvement in reproductive performance may be due to changes in the structure of ASMT protein. For g.7979477C>T of ADAMTS1 gene, Jining Grey does with genotype CT or TT had 0.82 (p < 0.05) or 0.86 (p < 0.05) more kids than those with genotype CC. No significant difference (p > 0.05) was found in litter size between CT or TT genotypes. These results preliminarily indicated that C allele (g.158122T>C) of ASMT gene and T allele (g.7979477C>T) of ADAMTS1 gene are potential molecular markers which could improve litter size of Jining Grey goats and be used in goat breeding.
Asunto(s)
Proteína ADAMTS1/genética , Acetilserotonina O-Metiltransferasa/genética , Cabras/fisiología , Tamaño de la Camada/genética , Polimorfismo de Nucleótido Simple , Proteína ADAMTS1/metabolismo , Acetilserotonina O-Metiltransferasa/metabolismo , Animales , Femenino , Cabras/genética , Reacción en Cadena de la Polimerasa/veterinaria , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
A disintegrin and metalloprotease with thrombospondin motifs (ADAMTS) family are widely implicated in tissue remodeling events manifested in cancer development. ADAMTS1, the most fully characterized ADAMTS, plays conflicting roles in different cancer types; however, the role of ADAMTS1 in renal cell carcinoma (RCC) remains unclear. Herein, we found that ADAMTS1 is highly expressed in RCC tissues compared to normal renal tissues, and its expression was correlated with an advanced stage and a poor prognosis of RCC patients. In vitro, we observed higher expression of ADAMTS1 in metastatic (m)RCC cells compared to primary cells, and manipulation of ADAMTS1 expression affected cell invasion and clonogenicity. Results from protease array showed that ADAMTS1 is modulated by melatonin through mechanisms independent of the MT1 receptor in mRCC cells, and overexpression of ADAMTS1 relieved the invasion/clonogenicity and growth/metastasis inhibition imposed by melatonin treatment in vitro and in an orthotopic xenograft model. The human microRNA (miR) OneArray showed that miR-181d and miR-let-7f were induced by melatonin and, respectively, targeted the 3'-UTR and non-3'-UTR of ADAMTS1 to suppress its expression and mRCC invasive ability. Clinically, RCC patients with high levels of miR-181d or miR-let-7f and a low level of ADAMTS1 had the most favorable prognoses. In addition, ubiquitin/proteasome-mediated degradation of ADAMTS1 can also be triggered by melatonin. Together, our study indicates that ADAMTS1 may be a useful biomarker for predicting RCC progression. The novel convergence between melatonin and ADAMTS1 post-transcriptional and post-translational regulation provides new insights into the role of melatonin-induced molecular regulation in suppressing RCC progression.
Asunto(s)
Proteína ADAMTS1/metabolismo , Carcinogénesis/metabolismo , Carcinoma de Células Renales/metabolismo , Neoplasias Renales/metabolismo , Melatonina/farmacología , Proteínas de Neoplasias/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteína ADAMTS1/genética , Animales , Carcinogénesis/efectos de los fármacos , Carcinogénesis/genética , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Humanos , Neoplasias Renales/genética , Neoplasias Renales/patología , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Metástasis de la Neoplasia , Proteínas de Neoplasias/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Granulosa cells (GCs) are essential components of follicles and play a role in regulating follicle development. The aim of this study was to investigate certain cellular components involved in the proliferation, differentiation and functional characteristics of granulosa cells in the success of fertilization of human oocytes during invitro fertilization (IVF) via immunohistochemical techniques. In this study, 30 patients who were diagnosed as primary or secondary infertility, polycystic ovary syndrome in the IVF center of Memorial Hospital, Department of Obstetrics and Gynecology were included. The amount of Anti Müllerian Hormone (AMH) in blood and granulosa cell diameter and cell core diameter were measured in 20 cells collected from each patient. In addition, degeneration scoring and BAX, ADAMTS-1, IL-10 expressions in granulosa cells were evaluated (p <0.01). It was thought that apoptosis induced by human GCs might be an indicator of egg quality. Moderate expression of ADAMTS-1 was thought to be related to failure of ovulation, deterioration of oocyte quality and decreased fertilization rate. This decrease in AMH levels may be associated with defects in granulosa cells. Therefore, significantly lower AMH secretion and increase in IL10 expression levels in healthy people can be explained by the increase of granulocyte cells.
Las células de la granulosa (GC) son componentes esenciales de los folículos y tienen un papel en la regulación del desarrollo de éste. El objetivo del estudio fue investigar ciertos componentes celulares involucrados en la proliferación, diferenciación y características funcionales de las células de la granulosa en el éxito de la fertilización de los ovocitos humanos durante la fertilización in vitro (FIV) a través de técnicas inmunohistoquímicas. En este estudio, se incluyeron 30 pacientes diagnosticados con infertilidad primaria o secundaria, síndrome de ovario poliquístico en el centro de FIV del Departamento de Obstetricia y Ginecología del Hospital Memorial. La cantidad de Hormona Anti Mülleriana (AMH) en la sangre, el diámetro de las células de la granulosa y el diámetro del núcleo celular se midieron en 20 células obtenidas de cada paciente. Además, se evaluó la puntuación de degeneración y las expresiones BAX, ADAMTS-1, IL-10 en células de granulosa (p <0,01). Se estimó que la apoptosis inducida por los GC humanos podría ser un indicador de la calidad del huevo. Se estimó que la expresión moderada de ADAMTS-1 estaba relacionada con el fracaso de la ovulación, el deterioro de la calidad de los ovocitos y la disminución de la tasa de fertilización. La disminución en los niveles de AMH puede estar asociada con defectos en las células de la granulosa. Por lo tanto, el aumento de las células de granulocitos puede explicar la disminución significativa de la secreción de AMH y el aumento de los niveles de expresión de IL10 en personas sanas.
Asunto(s)
Humanos , Femenino , Fertilización In Vitro/métodos , Interleucina-10/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Proteína ADAMTS1/metabolismo , Células de la Granulosa/metabolismo , InmunohistoquímicaRESUMEN
Esophageal cancer is one of the most lethal malignancies worldwide, and esophageal squamous cell carcinoma (ESCC) is the dominant histological type. However, the long noncoding RNA (lncRNA) alterations in ESCC have not been elucidated to date. In this study, reliable databases from Gene Expression Omnibus (GEO), which analyzed lncRNA expression in ESCC tumor tissues and adjacent normal tissues were searched, and common differentially expressed lncRNAs and genes were analyzed. Next, cis- trans analysis was performed to predict the underlying relationships between altered lncRNAs and mRNAs, and the lncRNA-mRNA regulatory network was established. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of altered lncRNA-related genes were performed. The promising lncRNA HCG22 was validated by quantitative polymerase chain reaction (qPCR), and clinicopathological data were collected to identify the relationship between lncRNA HCG22 expression level and clinical features. Finally, Transwell assays were performed to explore the biological functions of lncRNA HCG22 in ESCC cells. Two hundred forty-one lncRNAs and 835 mRNAs were observed to be remarkably altered between ESCC tumor tissues and adjacent normal tissues. The lncRNA-mRNA regulatory network showed the coexpression association between lncRNA HCG22 and SPINK7 and ADAMTS12. GO and KEGG analyses showed that HCG22 and ADAMTS12 had potential biological functions in the cell migration of ESCC. The downregulation of lncRNA HCG22 in ESCC tumor tissues was validated by qPCR, and the clinicopathological data showed a noticeable correlation between lncRNA HCG22 expression level and the ESCC differentiational degree and clinical TNM stage. Kaplan-Meier analysis showed that patients with ESCC having low lncRNA HCG22 expression in ESCC tissues had considerably shorter overall survival compared with patients with ESCC having high lncRNA HCG22 expression. Following Transwell assays confirmed the migratory role of lncRNA HCG22 in ESCC cells. In conclusion, lncRNA HCG22 was downregulated in ESCC tissues and can be a migration inhibitor of ESCC cells, and SPINK7 and ADAMTS12 are promising to be the regulatory targets of lncRNA HCG22.
Asunto(s)
Proteína ADAMTS1/metabolismo , Movimiento Celular , Biología Computacional/métodos , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago/patología , ARN Largo no Codificante/genética , Inhibidores de Serinpeptidasas Tipo Kazal/metabolismo , Proteína ADAMTS1/genética , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Proliferación Celular , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas de Esófago/genética , Carcinoma de Células Escamosas de Esófago/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Pronóstico , Inhibidores de Serinpeptidasas Tipo Kazal/genética , Tasa de Supervivencia , Células Tumorales CultivadasRESUMEN
MicroRNAs (miRNAs) have attracted increasing attention for their function in oocyte in vitro maturation (IVM). In this study, we aimed to explore the functional role and underlying mechanism of miR-375 in oocyte IVM. Cumulus-oocyte complexes (COCs) were cultured in standard cell culture conditions until they reach metaphase II (MII) stage. MiR-375 overexpression or knockdown was achieved by corresponding lentiviral transduction. Levels of miR-375, disintegrin and metalloproteinase with thrombospondin-like motifs 1 (ADAMTS1) mRNA and progesterone receptor (PGR) mRNA were detected by qRT-PCR. Western blotting was used to assess the expression of ADAMTS1 and PGR protein. The targeted interaction between miR-375 and ADAMTS1 or PGR was verified by dual-luciferase reporter assay and RNA immunoprecipitation assay. Our results demonstrate that miR-375 is downregulated, and ADAMTS1 and PGR are upregulated in cumulus cells during COC maturation. MiR-375 negatively regulates COC maturation. Moreover, ADAMTS1 and PGR are two targets of miR-375 in cumulus cells. ADAMTS1 or PGR knockdown represses COC maturation and miR-375 inhibits the expression levels of ADAMTS1 and PGR in cumulus cells. Additionally, miR-375 overexpression-mediated suppressive effect on COC maturation is abated by ADAMTS1 or PGR expression restoration. In conclusion, our study suggests that miR-375 represses oocyte IVM at least partially through targeting ADAMTS1 and PGR in cumulus cells, providing a novel insight for the involvement and underlying mechanism of miR-375 in oocyte IVM.